PARATOPE CHARACTERIZATION BY STRUCTURAL MODELING OF 2 ANTI-CORTISOL SINGLE-CHAIN VARIABLE FRAGMENTS PRODUCED IN ESCHERICHIA-COLI

Two monoclonal antibodies (mAbs), 5A4 and 6D6, directed against cortis ol, have been obtained; 6D6 is used in an assay kit for cortisol. The antibodies also recognize other, structurally related steroids present in the sample assayed. To improve the specificity of the assay, we ai med to minimize the recognition of non-cortisol steroids by the two an ti-cortisol mAbs. Our strategy consisted in constructing an efficient expression vector in E. coli which produced the single-chain variable fragment (scFv) of the mAbs in the periplasmic space. We demonstrated that temperature and inducer concentration of the bacterial culture in fluenced dramatically the yield of active scFv. From the nucleotide se quence we constructed a three-dimensional model of the two variable fr agments in order to understand why related steroids are, or are not re cognized by the antibody. For both antibodies, we have identified chem ical groups which are probably involved in the binding of the steroid haptens and the antibodies. The hydrophobic pocket formed by the antib ody comprises two or three tryptophan residues which can interact with the steroid nucleus by stacking. The serine at position 35 of the hea vy chain is buried in the back of the pocket and can form a hydrogen b ond with the 20-keto group of the cortisol. The stacking interactions and the hydrogen bond orient the steroid in the pocket. This reactivit y of the binding site is sustained by the analysis of the cross-reacti ons of related steroids with the mAbs.